(N=4 mice/group). treatment with ASHMI? and did not re-occur following OVA re-challenge up to 8 wks post-therapy. Reduced allergen-specific IgE and Th2 cytokine levels, and increased IFN- levels also persisted at least 8 wks post-therapy. ASHMI? effects were eliminated by neutralization of IFN-, but not TGF-, during therapy. Conclusion ASHMI? induced long-lasting post-therapy tolerance to antigen-induced inflammation and AHR. IFN- is a critical factor in ASHMI? effects. (Ling-Zhi), Ait (Ku-Shen), Fischer (Gan-Gao). ASHMI? was prepared by the Sino-Lion Pharmaceutical Organization ( a GMP qualified facility) Weifang, China) as described previously .Composition, quality control and chemistry analysis have been described in extensive detail previously[16;17]. Endotoxin levels in ASHMI? were tested using the Pyrogent Plus assay kit (Lonza, MA). No endotoxin was detected in ASHMI? [ 0.03 EU/ml, the limit of sensitivity for this kit]. Protocol for induction of asthma, ASHMI? treatment and post therapy evaluation Female 6-week- aged BALB/c (Jackson Laboratory, ME) mice sensitized twice with weekly intraperitoneal (i.p.) injections of 200g, ovalbumin (OVA, Type V; Sigma-Aldrich, , MO) and 2 mg of alum (Thermo Scientific, IL) in 0.4 ml of phosphate buffered saline (PBS), were challenged intratracheally (i.t.) with 100 g OVA in 50 l PBS weekly for 3 weeks (Fig 1A). Forty-eight hours after the third challenge, airway pressure time index (APTI) measurements and bronchoalveolar lavage were performed in a group of Adenine sulfate OVA mice compared to na?ve mice (n=5 per group) Additional groups of OVA mice were given 9 mg ASHMI? in 0.5 ml of drinking water twice daily (OVA/ASHMI?) or water (OVA/Sham) by oral gavage for 4 weeks. The ASHMI? dose was determined by a conversion table of equivalent human to animal dose ratios based on body surface area. Na?ve mice served as normal controls. Post-therapy evaluations for AHR and airway inflammation were performed at 1 day, Adenine sulfate 4 weeks and 8 weeks post therapy Hpt on individual groups of mice (Fig 1A). Open in a separate window Physique 1 A-Protocol: Mice were sensitized and challenged with OVA at times indicated. Daily oral ASHMI? treatment lasted for 4 weeks. Mice were subsequently challenged 1 day-, 4- and 8 weeks post therapy.. B-AHR (left panel) and % of BAL eosinophils (right panel) were measured 48 hrs after 5th OVA challenge (day 58) and the 9th OVA challenge (day 113). Data shown as Mean SD in individual samples (N=4C5 mice/group). *, values 0.05 were considered significant. RESULTS ASHMI? produced long-term post therapy suppression of allergic airway responses ASHMI? treatment commenced after Adenine sulfate the 3rd OVA challenge (Fig 1A). At this time asthmatic responses were fully established (Supplementary Physique 2). Immediately after completing treatment, OVA/sham mice showed sustained AHR following OVA re-challenge, evidenced by significantly higher APTI levels than na?ve mice (p 0.001, Fig 1 B, left panel). In contrast, OVA/ASHMI? mice showed complete resolution of AHR evidenced by Adenine sulfate normal APTI levels. Importantly, OVA/ASHMI? mice showed no AHR in response to subsequent OVA re-challenges up to 8 wks post-therapy, whereas OVA/Sham mice showed sustained AHR. Consistently, immediately after completing treatment, eosinophil figures were significantly reduced in OVA/ASHMI? mice as compared to the OVA/Sham mice (P 0.01C0.001, Fig 1B, right panel), and similar results were observed 8 weeks post-therapy 48 hours following the final antigen challenge (P 0.01C0.001 vs sham). Histological analysis also showed near normal features in OVA/ASHMI? mice, much like na?ve mice immediately (data not shown) and 8 weeks post-therapy (Fig 1C). Consistent with the BAL data, OVA/Sham mice exhibited pronounced peribronchial inflammation containing numerous eosinophils (Fig 1 C, panel i and indicated by arrows in inset). As illustrated in Fig 1 C panel ii, inflammation was dramatically reduced in the lungs from OVA/ASHMI? mice. Numerous goblet cells were present.